Integrase (IN) is encoded by the Pol gene from the HIV provirus. Our laboratory can efficiently express IN as an active recombinant protein, and has pioneered the integrase inhibitors research field (our landmark publication: PNAS 1993), discovered several families of lead inhibitors, demonstrated that IN inhibitors act as interfacial inhibitors (Nature Rev Drug Discovery 2012), and been granted several patents for IN inhibitors for therapeutic development. Our current studies are focused on the optimization of our novel chemotype integrase inhibitors to overcome resistance to raltegravir, elvitegravir and dolutegravir and target novel sites of IN. We have published and patented novel synthetic chemotypes as IN strand transfer inhibitors (INSTIs) including phtalimide and quinolinonyl derivatives in collaborations with Dr. Terrence Burke, Chemical Biology Laboratory (CCR, NCI). We have developed a panel of recombinant IN proteins bearing the mutations observed in patients that develop resistance to raltegravir, elvitegravir and dolutegravir. Using our resistant IN mutants, we have characterized the molecular pharmacology of elvitegravir, dolutegravir and our novel inhibitors, comparing them to raltegravir. We have shown that raltegravir, elvitegravir, dolutegravir and our novel series are highly selective for the strand transfer reaction, while being more than 100-fold less potent against the 3'-processing reaction, and almost inactive against the disintegration reaction mediated by integrase. The selective activity against strand transfer (one of the 3 reactions mediated by integrase) demonstrates the very high specificity of the clinically developed IN strand transfer inhibitors (INSTIs). It is consistent with our pharmacological hypothesis (Nature Drug Discovery 2012) that the strand transfer inhibitors trap the IN-viral DNA complex by chelating the divalent metals in the enzyme catalytic site following 3'-processing of the viral DNA and with our co-crystal structure and molecular modeling data. We have characterized the biochemical enzymatic activities and drug sensitivities of the IN mutants that confer clinical drug resistance. We have expanded these studies to double-mutants in the integrase flexible loop that commonly arise in raltegravir-resistant patients. Our results support the value of dolutegravir to overcome resistance to raltegravir and elvitegravir and facilitate patient compliance. We have determined additional crystal structures of wild-type and mutant prototype foamy virus (PFV) intasomes bound to our new series of inhibitors in collaboration with Dr. Peter Cherepanov at the Crick Institute, Cancer UK Center in London. The ability to structurally adapt to the structural changes associated with drug resistance is now achievable to rationally develop our new INSTIs. This year, we have also performed biochemical experiments demonstrating the feasibility of designing peptide inhibitors of IN. Because our XZ compounds possess high therapeutic index without cytotoxicity up to 250 microM (the highest dose tested), 3 of them, XZ419, XZ434 and XZ446 have been selected for preclinical development and animal testing by the NCI-CCR Drug Development Collaborative group (DDC). It is likely that further chemical modifications will be necessary to optimize formulation and pharmacokinetics. IATAP support will allow us to continue our collaborative work with Terrence Burke and XueZhi Zhao and with Stephen Hughes to achieve these goals.